Generation of serum-free media for expansion of human T lymphocytes and CD34+ HSCs using High-Dimensional Design-of-Experiments (HD-DoE) ABSTRACT Serum-containing media, which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte and hematopoietic stem cell cultures. Serum supplements available in the market such as Human Platelet Lysate (HPL) is not a solution, as their composition remains highly variable with a mix of growth factors, miRNAs, microvesicles, exosomes and thus difficult to standardize due to significant batch to batch variation. Although clinical trials are ongoing with T cells expanded in commercially available serum-free media, they are obliged to be supplemented with human Type AB serum to facilitate optimal growth and T cell activation. By using Trailhead Biosystems' QbD approach on primary human bone marrow isolates and a novel clustering algorithm developed by the company, we previously mapped the fate space of early hematopoiesis and identified completely novel set of input factors related to long-term marrow populating cells (LT-HSC). Given the importance of advanced cell culture systems for successful manufacturing of T cells and HSCs therapies, this project further aims to determine optimal conditions (most effective effector combinations and concentrations) to develop serum-free, chemically defined media formulations by using high- dimensional DoE designs for pathway interrogation along with Multivariate Data Analysis (MVDA). These novel media will be validated for their proliferative response on activation, phenotype, and functionality comparatively against serum substituted media and ?serum alternative? commercially available media. This project will deliver two optimized media formulations: T-SFMOPT and HSC- SFMOPT each specific to serum free expansion of T cell and HSCs, respectively. The commercial and clinical impact of the project rests on improved and successful clinical outcomes of transplanted T cells and LT-HSCs expanded in serum free media for treating life threatening hematological and immunological diseases.